PROJECT SUMMARY/ABSTRACT The exclusively blood-feeding blacklegged tick Ixodes scapularis is the most medically relevant vector of infectious diseases in the United States. Despite the public health importance of I. scapularis, which transmits over six pathogens, including the Lyme disease spirochete Borrelia burgdorferi, this non-insect arthropod vector is understudied. The molecular details of vector-pathogen interactions and non-insect arthropod immune responses remain relatively poorly characterized. The recent description of the tick immune deficiency (IMD) pathway elucidated one facet of the tick response to B. burgdorferi. The IMD pathway is centrally regulated by the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP), which serves to polyubiquitylate target proteins, forming ubiquitin scaffolds. Substrates of XIAP in the context of tick-B. burgdorferi interactions remain unknown. In this F31 application, we report an interacting partner, p47, which is shown to participate in the tick immune response to B. burgdorferi by restricting pathogen acquisition in I. scapularis. The central hypothesis of the proposed research is that XIAP binds and polyubiquitylates p47 in a lysine (K)63-dependent manner. We will investigate this hypothesis in two aims. In Aim 1, the molecular and biophysical details of the XIAP-p47 interaction will be systematically examined by using p47 deletion mutants in binding assays with XIAP, as well as surface plasmon resonance and isothermal titration calorimetry. In Aim 2, biochemical interactions between XIAP and p47 will be investigated by performing in vitro polyubiquitylation assays and mass spectrometry to identify which residues of p47 are polyubiquitylated. By completing the aims of the proposed research, we will characterize a substrate of XIAP, which will improve our understanding of the tick IMD pathway. These findings will also contribute to unraveling novel scientific paradigms in tick-pathogen interactions.